Investigating the Integrator Complex's Role in Regulating Inflammatory Transcription

研究整合复合体在调节炎症转录中的作用

• 批准号: 10536610
• 负责人: Chad Brandon Stein
• 金额: $ 1.74万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-01-01 至 2023-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10536610
• 关键词: Address; Affect; Autoimmune Diseases; Biological Models; Bone Marrow; Cell surface; Cells; Chronic; Clinical; Code; Complex; Crohn' s disease; Cues; DNA; Data; Data Set; Development; Disease; Dissociation; Drosophila genus; Endoribonucleases; Equilibrium; Gene Activation; Genes; Genetic Transcription; Genomics; Homeostasis; Hour; Human; Human Genetics; Immune; Immune response; Immune system; Immunoprecipitation; Inflammatory; Inflammatory Bowel Diseases; Inflammatory Response; Innate Immune System; Interphase Cell; Invaded; Literature; Location; Macrophage; Mass Spectrum Analysis; Mediating; Messenger RNA; Modeling; Molecular; Monitor; Morbidity - disease rate; Mus; Nucleotides; Organism; Pathogenesis; Phosphotransferases; Physiological; Physiology; Play; Polymerase; Process; Productivity; Property; Proteins; RNA; RNA Binding; RNA Polymerase II; RNA chemical synthesis; Reaction; Regulation; Repression; Rest; Ribonucleosides; Risk; Role; Route; Signal Transduction; Site; Stimulus; Structure; Systemic infection; Therapeutic; Transcript; Transcription Initiation Site; Transcriptional Regulation; Transgenes; Ulcerative Colitis; Up-Regulation; Work; cell type; crosslink; cytokine; derepression; embryonic stem cell; endonuclease; experimental study; gene repression; insight; monocyte; pathogen; prevent; programs; promoter; protein complex; recruit; response; small molecule; therapeutic target; transcription termination; transcriptome sequencing

Inflammatory diseases like Ulcerative Colitis and Crohn’s Disease are increasingly prevalent chronic conditions with significant morbidity if untreated. While the exact molecular underpinnings of these diseases are unknown, it is clear that misregulation of the innate immune system plays a key role in their pathogenesis. Importantly, precise control of mRNA synthesis by RNA Polymerase II (RNAPII) is essential for immune homeostasis and responses to environmental cues like invading pathogens. In metazoans, RNAPII pauses shortly downstream of transcription start sites, where the polymerase can remain in a poised state until transcriptional kinases release it into productive elongation. Promoter-proximal pausing is widespread and is critical for proper responses to stimuli like pro-inflammatory molecules. Recently, it has become appreciated that another fate for paused RNAPII is common: promoter-proximal termination. Here, instead of paused polymerase being released into productive elongation, it is dissociated from its DNA template and releases a short, non-functional mRNA. Promoter-proximal pausing is critical for proper regulation of pro-inflammatory genes, so understanding this process is important for human physiology and disease states. Our group and others recently showed that the Integrator complex is responsible for inducing promoter- proximal termination at a subset of protein-coding genes. The Integrator complex is essential for viability, comprised of at least 14 subunits, and induces termination through cleavage of nascent RNA. Specifically, the catalytically active Integrator Subunit 11 (INTS11) induces RNA cleavage. However, the regulation of Integrator’s catalytic activity is poorly understood. A growing body of work shows that Integrator regulates responses to pro- inflammatory cues, and human genetics suggests its involvement in inflammatory bowel disease pathogenesis. Here, I aim to uncover how Integrator activity is regulated, and how this complex affects transcription of inflammatory genes. Because of Integrator’s emerging role in regulating inflammatory transcription, I will use mouse embryonic stem cell-derived macrophages (ESDMs) as a model system. I will first determine Integrator’s role in inflammatory transcription by rapidly depleting INTS11 and monitoring cells’ ability to mount an immune response. Using rapid INTS11 depletion paired with nascent RNA sequencing, I will precisely define the targets of Integrator in this physiologically relevant immune cell type. I will also determine where Integrator is targeted and explore whether its genomic localization changes in response to immune challenge. Next, I will characterize the RNA-binding properties of Integrator in an effort to understand how its catalytic activity is regulated. Finally, I will probe Integrator protein partners, which will shed light on how it is targeted and how it interacts with the transcription machinery. Together, this work will provide insight into this understudied complex and may allow for Integrator to be therapeutically targeted in inflammatory disease.

溃疡性结肠炎和克罗恩病等炎症性疾病越来越普遍 如果未经治疗，发病率明显。尽管这些疾病的确切分子基础尚不清楚，但 显然，对先天免疫系统的不利地位在其发病机理中起关键作用。重要的是， RNA聚合酶II（RNAPII）对mRNA合成的精确控制对于免疫稳态和 对环境线索的反应，例如入侵病原体。在后生动物中，RNAPII很快就会下游 转录起始位点，聚合酶可以保持中毒状态，直到转录激酶释放 它变成生产性延伸。促进者暂停是广泛的，对于适当的回应至关重要 像促炎分子一样刺激。最近，人们对暂停的另一个命运已被赞赏 RNAPII是常见的：启动子终止。在这里，而不是暂停的聚合酶被释放到 生产性伸长，它与其DNA模板分离，并释放出短而非功能的mRNA。 启动子 - 抗性暂停对于适当调节促炎基因至关重要，因此请理解这一点 过程对于人类生理和疾病状态很重要。 我们的小组和其他人最近表明，集成仪综合体负责诱导的启动子 - 蛋白质编码基因子集的近端终止。集成仪复合物对于可行性至关重要， 完成至少14个亚基，并通过新生RNA的裂解诱导终止。具体来说， 催化活性积分子亚基11（INTS11）诱导RNA裂解。但是，集成商的调节 催化活性知之甚少。越来越多的工作表明，集成商调节对计划的反应 炎症提示，人类遗传学表明它参与了炎症性肠病发病机理。 在这里，我的目的是揭示集成剂活动的调节方式，以及该复合物如何影响 炎症基因。 由于积分器在调节炎症转录中的新兴作用，我将使用鼠标胚胎 干细胞衍生的巨噬细胞（ESDMS）作为模型系统。我将首先确定集成商在 通过快速耗尽INTS11并监测细胞安装免疫的能力，炎症转录 回复。使用快速INTS11部署与新生的RNA测序配对，我将精确定义目标 在这种物理相关的免疫细胞类型中积分器的。我还将确定集成符的目标 并探索其基因组定位是否会响应免疫挑战而发生变化。接下来，我将描述 积分器的RNA结合特性是为了了解其催化活性如何受到调节。最后， 我将探测积分蛋白合作伙伴，该伙伴将阐明其目标的方式以及它如何与之相互作用 转录机械。这项工作将共同洞悉这种理解的复杂，并可能允许 使整合物成为炎症性疾病中的热靶向。